Isolation of atypical enteropathogenic Escherichia coli from chicken and chicken-derived products.

Atypical enteropathogenic Escherichia coli (EPEC) strains from chicken and chicken-derived products were isolated and characterised. The strains presented a wide variety of serotypes, some have been reported in other animal species (O2:H40, O5:H40) and in children with diarrhoea (O8:H-). Most of the strains carried intimin ß. The results indicate that chicken and chicken products are important sources of atypical EPEC strains that could be associated with human disease, and highlight the need to improve hygiene practices in chicken slaughtering and meat handling.

Virulence genotypes, antibiotic resistance and the phylogenetic background of extraintestinal pathogenic Escherichia coli isolated from urinary tract infections of dogs and cats in Brazil.

Urinary tract infection (UTI) is a frequent disease of humans and pets and has extra-intestinal pathogenic Escherichia coli (ExPEC) strains as one of the main etiologic agent. ExPEC are characterized by specific virulence factors and are related to a heterogeneous group of human and animal disorders, besides to be a relevant participant in the dissemination of antimicrobial resistance. The purpose of this study was to characterize E. coli strains isolated from UTI of dogs and cats for serotypes, virulence markers, phylogenetic groups and sensitivity to antimicrobial drugs. E. coli was identified as the etiologic agent of UTI in urine samples of 43 pets (7 cats and 36 dogs). Serogroups O2, O4 and O6 corresponded to more than one third of the isolates, being 62% of the total strains classified as B2, 18% as D, 16% as B1 and 4% as A. The iucD (22%), fyuA (80%), traT (51%) and cvaC (20%) genes were distributed among the four phylogenetic groups, whereas the papC/papEF (47%) and malX (67%) genes were found only in groups B2 and D. There were a high number of resistant strains, with 76% of the strains belonging to groups A, B1 and D characterized as multidrug resistant (MDR), whereas only 21% had this phenotype in the group B2. The ExPEC strains isolated in this study displayed pathotypic and phylogenetic similarities with human isolates and high percentages of drug resistance. The finding of MDR ExPEC strains suggests implications for animal and public health and deserves more investigations.

Shiga toxin-producing Escherichia coli in drinking water supplies of north Paraná State, Brazil.

AIM: To determine the occurrence and characteristics of Shiga toxin-producing Escherichia coli (STEC) in drinking water supplies treated and untreated. METHODS AND RESULTS: Drinking water samples (n = 1850) were collected from 41 municipalities in the north of Paraná State between February 2005 and January 2006. Escherichia coli isolates (n = 300) were recovered from water and investigated for the presence of virulence markers related to STEC by PCR. STEC isolates recovered were then characterized for both phenotypic and genotypic traits. A total of 12 isolates (11 from untreated water and one from treated water) were positive for stx, including five positive for both stx1 and stx2, two positive for stx1 and five positive for stx2. None of the STEC isolates contained eae, but other virulence genes were observed such as ehxA (100%), saa (100%), lpfAO113 (75%), iha (42%), subAB (25%) and cdtV (8%). Multidrug resistance was identified in 25% of the STEC isolates. The 12 STEC isolates belonged to seven distinct serotypes and pulsed-field gel electrophoresis typing revealed the presence of two clusters and two clones in this region. CONCLUSION: Drinking water, especially from untreated water supplies, can be source of STEC strains potentially pathogenic for humans. SIGNIFICANCE AND IMPACT OF THE STUDY: The investigation of the drinking water supplies for pathogenic E. coli, as STEC, may be useful to prevent waterborne outbreaks.

Genotypic characterization of non-O157 Shiga toxin-producing Escherichia coli in beef abattoirs of Argentina.

The non-O157 Shiga toxin-producing Escherichia coli (STEC) contamination in carcasses and feces of 811 bovines in nine beef abattoirs from Argentina was analyzed during a period of 17 months. The feces of 181 (22.3%) bovines were positive for non-O157 STEC, while 73 (9.0%) of the carcasses showed non-O157 STEC contamination. Non-O157 STEC strains isolated from feces (227) and carcasses (80) were characterized. The main serotypes identified were O178:H19, O8:H19, O130:H11, and O113:H21, all of which have produced sporadic cases of hemolytic-uremic syndrome in Argentina and worldwide. Twenty-two (7.2%) strains carried a fully virulent stx/eae/ehxA genotype. Among them, strains of serotypes O103:[H2], O145:NM, and O111:NM represented 4.8% of the isolates. Xba I pulsed-field gel electrophoresis pattern analysis showed 234 different patterns, with 76 strains grouped in 30 clusters. Nine of the clusters grouped strains isolated from feces and from carcasses of the same or different bovines in a lot, while three clusters were comprised of strains distributed in more than one abattoir. Patterns AREXSX01.0157, AREXBX01.0015, and AREXPX01.0013 were identified as 100% compatible with the patterns of one strain isolated from a hemolytic-uremic syndrome case and two strains previously isolated from beef medallions, included in the Argentine PulseNet Database. In this survey, 4.8% (39 of 811) of the bovine carcasses appeared to be contaminated with nonO157 STEC strains potentially capable of producing sporadic human disease, and a lower proportion (0.25%) with strains able to produce outbreaks of severe disease.

Characterization of Shiga toxin-producing Escherichia coli isolated from dairy cows in Argentina.

AIMS: To feno-genotypically characterize the Shiga toxin-producing Escherichia coli (STEC) population in Argentinean dairy cows. METHODS AND RESULTS: From 540 STEC positive samples, 170 isolates were analyzed by multiplex PCR and serotyping. Of these, 11% carried stx1, 52% stx2 and 37% stx1/stx2. The ehxA, saa and eae were detected in 77%, 66% and 3%, respectively. Thirty-five per cent of strains harboured the profile stx1, stx2, saa, ehxA and 29% stx2, saa, ehxA. One hundred and fifty-six strains were associated with 29 different O serogroups, and 19 H antigens were distributed among 157 strains. STEC O113:H21, O130:H11 and O178:H19 were the most frequently found serotypes. The STEC O157:H7 were detected in low rate and corresponded to the stx2(+) , eae(+) , ehxA(+) virulence pattern. CONCLUSIONS: We detected a diversity of STEC strains in dairy cattle from Argentina, most of them carrying genes linked to human disease. SIGNIFICANCE AND IMPACT OF THE STUDY: The non-O157 STEC serotypes described in this study are associated worldwide with disease in humans and represent a risk for the public health. For this, any microbiological control in dairy farms should be targeted not only to the search of O157:H7 serotype.

Urinary tract infections in renal transplant recipients: virulence traits of uropathogenic Escherichia coli.

BACKGROUND: Urinary tract infection (UTI) is the most common infectious complication after renal transplantation. Most infections are caused by uropathogenic Escherichia coli (UPEC). There are limited data on the prevalence of virulence traits among UPEC isolated from renal transplant recipients. This study compared the phenotypic and genotypic profiles of UPEC strains isolated from recipients with those from control patients. METHODS: E coli isolates that caused UTI in recipients versus nonimmunosuppressed control patients were characterized according to phylogenetic group and the presence of urovirulence genes pap1/pap2; sfa1/sfa2; afa1/afa2; aer1/aer2; and cnf1/cnf2. RESULTS: Thirty-six UPEC isolates from recipients and another 27 from control individuals were included in the study. The proportion of episodes of pyelonephritis in recipients (50%) versus control subjects (41%) was similar (P = .46). However, secondary bacteremia was observed only among recipients (n = 8; P < .001). There was no significant difference in the distribution of phylogenetic groups or the prevalence of analyzed virulence traits between UPEC isolated from the 2 groups. Nevertheless, strains associated with secondary bacteremia in recipients showed a higher prevalence of mannose-resistant hemagglutination (P = .013). CONCLUSION: The phenotypic and genotypic characteristics of UPEC isolated from recipients were similar to those from control patients at a tertiary care center. Secondary bacteremia in recipients was associated with a higher prevalence of mannose-resistant hemagglutination.

Shiga toxin-producing Escherichia coli and atypical enteropathogenic Escherichia coli strains isolated from healthy sheep of different populations in São Paulo, Brazil.

AIMS: Sheep are important carriers of Shiga toxin-producing Escherichia coli (STEC) in several countries. However, there are a few reports about ovine STEC in American continent. METHODS AND RESULTS: About 86 E. coli strains previously isolated from 172 healthy sheep from different farms were studied. PCR was used for detection of stx(1), stx(2), eae, ehxA and saa genes and for the identification of intimin subtypes. Restriction fragment length polymorphism (RFLP)-PCR was performed to investigate the variants of stx(1) and stx(2), and the flagellar antigen (fliC) genes in nonmotile isolates. Five isolates were eae(+) and stx(-), and belonged to serotypes O128:H2/beta-intimin (2), O145:H2/gamma, O153:H7/beta and O178:H7/epsilon. Eighty-one STEC isolates were recovered, and the stx genotypes identified were stx(1c)stx(2d-O118) (46.9%), stx(1c) (27.2%), stx(2d-O118) (23.4%), and stx(1c)stx(2dOX3a) (2.5%). Pulsed-field gel electrophoresis (PFGE) revealed 27 profiles among 53 STEC and atypical enteropathogenic Escherichia coli (EPEC) isolates. CONCLUSIONS: This study demonstrated that healthy sheep in São Paulo, Brazil, can be carriers of potential human pathogenic STEC and atypical EPEC. SIGNIFICANCE AND IMPACT OF THE STUDY: As some of the STEC serotypes presently found have been involved with haemolytic uraemic syndrome (HUS) in other countries, the important role of sheep as sources of STEC infection in our settings should not be disregarded.

A prospective study on Shiga toxin-producing Escherichia coli in children with diarrhea in Paraná State, Brazil.

AIMS: To examine stool specimens from children with diarrhea from Paraná State, southern Brazil, for presence of STEC. METHODS AND RESULTS: A PCR screening assay for stx genes was used to examine a loopful of confluent colonies of 306 stool samples cultures. In six (1.96%) of them, DNA fragments of the expected size were observed, and the presence of stx was confirmed by DNA sequencing. Then up to 100 single colonies from each of the six stool cultures were analyzed using the same PCR protocol. However, stx-positive colonies were found only in two of the cultures. The E. coli strains belonged to serotypes O69:H11 and O178:H19, and presented genotypes stx(1)eae ehxA and stx(1) respectively. Shiga toxin production was confirmed using the VTEC Screen Seiken. Except ampicillin, they were susceptible to all the antimicrobials tested. CONCLUSIONS: These results show that STEC may be an important cause of diarrhea in children of Paraná State, and that they are present in low numbers in stools. The strains belonged to serotypes not commonly found associated with STEC and probably present low virulence. SIGNIFICANCE AND IMPACT OF STUDY: These results indicate that molecular methods are required to diagnosis of STEC infections.

Phenotypic and genotypic traits of Shiga toxin-producing Escherichia coli strains isolated from beef cattle from Paraná State, southern Brazil.

AIMS: To investigate the prevalence and characteristics of Shiga toxin-producing Escherichia coli (STEC) in cattle from Paraná State, southern Brazil. METHODS AND RESULTS: One hundred and seven faeces cattle samples were cultured on Sorbitol-MacConkey agar. Escherichia coli colonies were tested for production of Shiga toxin using Vero-cell assay. A high prevalence (57%) of STEC was found. Sixty-four STEC were serotyped and examined for the presence of stx(1), stx(2), eae, ehxA and saa genes and stx(2) variants. The isolates belonged to 31 different serotypes, of which three (O152:H8, O175:H21 and O176:H18) had not previously been associated with STEC. A high prevalence of stx(2)-type genes was found (62 strains, 97%). Variant forms found were stx(2), stx(2c), stx(2vhb), stx(2vO111v/OX393) and a form nonclassifiable by PCR-RFLP. The commonest genotypes were stx(2)ehxA saa and stx(1)stx(2)ehxA saa. CONCLUSIONS: A high frequency of STEC was observed. Several strains belong to serotypes previously associated with human disease and carry stx(2) and other virulence factors, thus potentially representing a risk to human health. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study of STEC in Paraná State, and its findings emphasize the need for proper cattle handling to prevent human contamination.

Distinct pathotypes of O113 Escherichia coli strains isolated from humans and animals in Brazil.

Two distinct diarrheagenic Escherichia coli pathotypes, enteroaggregative E. coli (EAEC) and Shiga toxin-producing E. coli, were observed in association with O113 strains isolated from human and nonhuman sources in Brazil, respectively. The O113 strains from human diarrhea belonged to a diversity of serotypes, and nine (53%) of them harbored virulence traits of typical EAEC.

Virulence markers and serotypes of Shiga toxin-producing Escherichia coli, isolated from cattle in Rio Grande do Sul, Brazil.

AIMS: To determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) and serotypes and virulence markers of the STEC isolates from beef and dairy cattle in Rio Grande do Sul, Brazil. METHODS AND RESULTS: Faecal samples from beef cattle were collected at slaughterhouses. The isolates were submitted to colony hybridization assay with specific DNA probes for stx1, stx2 and eae genes, and serotyped for the identification of O and H antigens. Thirty-nine per cent of beef cattle surveyed harboured at least one STEC strain. Among the distinct serotypes identified, 10 were shared by both beef and dairy cattle. Most of the strains isolated harboured stx2. Genotypic and phenotypic profiles allowed the identification of 34 and 31 STEC strains, isolated from beef and dairy cattle, respectively. Serotypes O10:H14, O15:H21, O96:H21, O119:H4, O124:H11, O128:H21, O137:H-, O141:H19, O159:H42, O160:H2 and O177:H11, identified in this study, have not been previously reported as STEC isolated from cattle. CONCLUSIONS: Cattle are an important reservoir of STEC strains associated with human diseases in South America. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the prevalence, genotypic profile and serotypes of STEC strains isolated from cattle enables the prediction of possible risk for public health.

Shiga toxin-producing and attaching and effacing Escherichia coli in cats and dogs in a high hemolytic uremic syndrome incidence region in Argentina.

Shiga toxin-producing Escherichia coli (STEC), responsible for the hemolytic uremic syndrome, is an endemic pathogen in Argentina. We studied the prevalence of STEC in fecal samples from cats and dogs of Buenos Aires city and suburbs. Cultures were used for screening stx1/stx2 and rfbO157 by multiplex PCR. All E. coli-positive colonies for these genes were further characterized for the eae gene and for serotypes. In dogs, 17 (3.7%), 19 (4.2%) and 34 (7.5%) of samples were positive for stx2, stx1 and rfb, respectively. In cats, six (4.0%) of the samples were positive for stx2, three (2.0%) for stx1 and four (2.7%) for rfbO157. In 18 (4.0%) of the dog samples, a bacteriological diagnosis was obtained by isolation. The percentage of positive isolates corresponding to the rfbO157 and to the stx2 genotypes were 2.9% and 1.1%, respectively. In four of the cat samples, the bacteriological diagnosis for stx2 (2.6% prevalence of STEC) was confirmed. Although these data suggest that the high infection index of STEC in children in Argentina does not seem to be due mainly to the role of cats and dogs, there are some strains with virulence genes in common for humans and their domestic animals.

Random amplification of polymorphic DNA reveals clonal relationships among enteropathogenic Escherichia coli isolated from non-human primates and humans.

Enteropathogenic Escherichia coli (EPEC) strains are important agents of infantile diarrhea all over the world, gaining even greater importance in developing countries. EPEC have also been isolated from various animal species, but most isolates belong to serotypes that differ from those recovered from humans. However, it has been demonstrated that several isolates from non-human primates belong to the serogroups and/or serotypes related to those implicated in human disease. The objective of this study was to evaluate the genetic differences between thirteen strains isolated from non-human primates and the same number of strains isolated from human infections. Human isolates belonged to the same serogroup/serotype as the monkey strains and the evaluation was done by analysis of random amplified polymorphic DNA. Dendrogram analysis showed that there was no clustering between human and monkey strains. Human and non-human isolates of the EPEC serotypes O127:H40 and O128:H2 shared 90 and 87% of their bands, respectively, indicating strong genomic similarity between the strains, leading to the speculation that they may have arisen from the same pathogenic clone. To our knowledge, this study is the first one comparing genomic similarity between human and non-human primate strains and the results provide further evidence that monkey EPEC strains correlate with human EPEC, as suggested in a previous investigation.

Random amplification of polymorphic DNA reveals clonal relationships among enteropathogenic Escherichia coli isolated from non-human primates and humans

Enteropathogenic Escherichia coli (EPEC) strains are important agents of infantile diarrhea all over the world, gaining even greater importance in developing countries. EPEC have also been isolated from various animal species, but most isolates belong to serotypes that differ from those recovered from humans. However, it has been demonstrated that several isolates from non-human primates belong to the serogroups and/or serotypes related to those implicated in human disease. The objective of this study was to evaluate the genetic differences between thirteen strains isolated from non-human primates and the same number of strains isolated from human infections. Human isolates belonged to the same serogroup/serotype as the monkey strains and the evaluation was done by analysis of random amplified polymorphic DNA. Dendrogram analysis showed that there was no clustering between human and monkey strains. Human and non-human isolates of the EPEC serotypes O127:H40 and O128:H2 shared 90 and 87 percent of their bands, respectively, indicating strong genomic similarity between the strains, leading to the speculation that they may have arisen from the same pathogenic clone. To our knowledge, this study is the first one comparing genomic similarity between human and non-human primate strains and the results provide further evidence that monkey EPEC strains correlate with human EPEC, as suggested in a previous investigation.

Characterization of Serratia marcescens isolates from subgingival biofilm, extraoral infections and environment by prodigiosin production, serotyping, and genotyping.

INTRODUCTION: Serratia marcescens is widely distributed in nature, and has emerged in the last years as an important nosocomial pathogen. The organism may also be found in subgingival biofilm in periodontitis patients. This study aimed to verify the subgingival prevalence of S. marcescens in different periodontal conditions and to evaluate whether the oral cavity would harbor strains similar to those causing infectious diseases. METHODS: The subgingival occurrence of S. marcescens was determined in 334 subjects. The phenotypic and genotypic diversity of 23 isolates from subgingival biofilm, 22 from extra-oral infections and 10 environmental strains, was compared by prodigiosin production, O and H serotyping and genotyping using polymorphic GC-rich repetitive sequences-polymerase chain reaction. RESULTS: S. marcescens was found more frequently in severe periodontitis patients (4.1%) than in gingivitis (3.2%) and healthy subjects (2.5%), but these differences were not statistically significant. Analysis of serotype distribution, prodigiosin production, and genotyping revealed that environmental strains were markedly different from most human isolates, either oral or extraoral. CONCLUSION: These data suggest that S. marcescens isolates from subgingival biofilm are not just contaminants from the environment, but that the oral cavity may act as a reservoir of strains able to promote human infections. However, further studies are needed to elucidate the role of this bacterium in the pathogenesis of periodontal diseases.

Serotypes and virulence markers of Shiga toxin-producing Escherichia coli (STEC) isolated from dairy cattle in São Paulo State, Brazil.

In order to determine the occurrence, serotypes and virulence markers of Shiga toxin-producing Escherichia coli (STEC) strains, 153 fecal samples of cattle randomly selected from six dairy farms in Sao Paulo State, Brazil, were examined for Shiga toxin (Stx) production by the Vero cell assay. Feces were directly streaked onto MacConkey Sorbitol Agar and incubated at 37 degrees C overnight. Sorbitol-negative colonies (maximum 20) and up to 10 sorbitol-positive colonies from each plate were subcultured onto presumptive diagnostic medium IAL. Sorbitol-negative isolates were screened with O157 antiserum for identification of O157:H7 E. coli. Isolates presenting cytotoxic activity were submitted to colony hybridization assays with specific DNA probes for stx1, stx2, eae, Ehly and astA genes. The isolation rate of STEC ranged from 3.8 to 84.6% depending on the farm analysed. STEC was identified in 25.5% of the animals, and most of them (64.1%) carried a single STEC serotype. A total of 202 STEC isolates were recovered from the animals, and except for the 2 O157:H7 isolates all the others expressed cytotoxic activity. The great majority of the STEC isolates carried both stx1 and stx2 genes (114/202, 56.4%) or stx2 (82/202, 40.6%); and whereas the Ehly sequence occurred in most of them (88%) eae was only observed in O157:H7 and O111:HNM isolates. Serotypes O113:H21, O178:H19 and O79:H14 were the most frequent STEC serotypes identified and widely distributed among animals from different farms, while others such as O77:H18, O88:H25 and O98:H17 occurred only in particular farms. This is the first report on the occurrence of STEC in dairy cattle in Sao Paulo State, and the results point to substantial differences in rate of isolation, serotypes and genetic profile of STEC that has been previously described among beef cattle in our community. Moreover, to our knowledge O79:H14 and O98:H17 represent new STEC serotypes, while O178:H19 has only been recently reported in Spain.

Attaching and effacing Escherichia coli isolated from dogs in Brazil: characteristics and serotypic relationship to human enteropathogenic E. coli (EPEC).

Escherichia coli isolates recovered from 182 fecal specimens from dogs up to five months old from the cities of São Paulo and Campinas, SP, Brazil, were examined by polymerase chain reaction (PCR) for several virulence factors and properties. The eae gene was found in 23 isolates of E. coli from 22 dogs, 19 of 146 (13%) from dogs with diarrhea and 3 of 36 (8.3%) from dogs with no diarrhea. Two different eae+ isolates were recovered from one dog with diarrhea. Isolates from two dogs with diarrhea harbored the bfpA gene, and none of the isolates possessed genes for enterotoxins, the EAF plasmid or Shiga toxins. PCR showed that, among the 23 isolates, eight were positive for beta intimin, six for gamma, two for, one for alpha, one for kappa, and five showed no amplification with any of the nine pairs of specific intimin primers used. PCR also showed that the LEE (locus of enterocyte effacement) was inserted in selC in four isolates, likely in pheU in seven isolates, and in undetermined sites in twelve isolates. Fifteen isolates adhered to HEp-2 cells and were fluorescence actin staining (FAS) positive. The predominant adherence pattern was the localized adherence-like (LAL) pattern. The eae-positive isolates belonged to a wide diversity of serotypes, including O111:H25, O119:H2 and O142:H6, which are serotypes that are common among human EPEC. These results confirmed the presence of EPEC in dogs (DEPEC) with and without diarrhea. The virulence factors found in these strains were similar to those in human EPEC, leading to the possibility that EPEC may move back and forth among human and canine populations.

Non-O157 Shiga toxin-producing Escherichia coli isolated from diarrhoeic calves in Argentina.

Faecal samples from 76 diarrhoeic calves belonging to 36 farms located in the Pampas plain, Argentina, were examined for Shiga toxin-producing Escherichia coli (STEC). A total of 15 STEC strains were isolated from 12 (15.8%) calves which came from six different farms. All stx positive strains assayed by PCR were also positives in the Vero cell cytotoxicity test. The majority (60.0%) of the STEC strains carried the stx(1) gene. Twelve (80.0%) of the STEC isolates which belonged to serotypes O5:H- (n = 4), O26:H11 (n = 4), O26:H- (n = 1), O111:H- (n = 2), and O123:H38 (n = 1) were also enterohaemolysin (EHly) positive and carried the gene encoding for intimin (eae). All the stx positive strains were negative for the bfpA gene. Localized adherence to HEp-2 cells were observed in 83.3% of the eae+ STEC strains. STEC belonging to serotype O5:H- showed atypical biochemical properties, including urease production. Urease was also produced by two strains belonging to serotypes O153:H? and non-typeable, respectively. Resistance to three or more antibiotics was observed in 12 (80.0%) of the STEC isolates. Most of the serotypes of STEC recovered in this survey carried virulence traits that are associated with increased human and bovine pathogenicity. The present study shows that highly virulent STEC strains are being shed by diarrhoeic calves from farms located in a high incidence area of human STEC infections.